A Novel Approach to Identify the Mediators in Chronic Itch

描述（由申请人提供）：慢性瘙痒是与皮肤，免疫和神经系统相关的许多疾病的表现。慢性瘙痒在很大程度上对传统的抗组胺药有耐药性，而且很少有有效的治疗方法。为了开发新的治疗慢性瘙痒的方法，迫切需要鉴定瘙痒特异性信号分子。我们最近发现了一小部分瘙痒特异性神经元，它们表达胃泌素释放肽受体（GRPR），这是脊髓中的一种瘙痒受体。GRPR+神经元是瘙痒性感知所必需的，但不是伤害性传递所必需的，这表明它们含有无数的瘙痒特异性基因。为了促进GRPR+神经元中新的瘙痒基因的鉴定，我们设计了一种新的策略，将GRPR+神经元中基因表达的全基因组分析与功能筛选相结合，包括行为测试和Ca2+成像。比较了正常GRPR+神经元与非正常GRPR+神经元浅层间的基因表达谱。这种策略使我们能够寻找在GRPR+神经元中表达的基因，这些基因在功能上是介导非组胺能性瘙痒所必需的。我们将进行三组实验来验证这一关键假设，即在脊髓中存在组胺能和非组胺能瘙痒的不同信号特征。首先，将通过微阵列、qRT-PCR和原位杂交研究对GRPR+神经元的基因表达进行全基因组分析。筛选GRPR+神经元中表达的候选基因。其次，利用siRNA敲低的方法评估候选基因在急性和慢性瘙痒中的作用，并确定急性或慢性瘙痒中介导非组胺能性瘙痒的关键基因。第三，候选基因与GRPR之间的关系将通过检测基因敲低是否会损害鞘内grp引起的抓伤，并进一步影响GRPR突变小鼠的抓伤行为来确定。此外，候选基因是否参与grpr依赖的Ca2+信号转导将在异种系统中使用siRNA敲除和Ca2+成像进行检查。拟议的研究具有高度的翻译性，因为瘙痒介质直接从慢性瘙痒模型中确定，并且可能是瘙痒特异性的。我们提出的研究将显著扩大未来药物发现计划可能探索的靶点池。

DESCRIPTION (provided by applicant): Chronic itch is a presenting sign in numerous diseases associated with the skin, the immune, and the nervous systems. Chronic itch is largely resistant to conventional antihistamines, and few effective therapies are available. In order to develop novel therapeutics against chronic itch, there is a pressing need for identification of itc-specific signaling molecules involved. We have recently uncovered a small subset of itch-specific neurons which express gastrin-releasing peptide receptor (GRPR), an itch receptor, in the spinal cord. GRPR+ neurons are required for pruritoceptive but not for nociceptive transmission, indicating that they contain a myriad of itch-specific genes. To facilitate the identification of novel itch genes in GRPR+ neurons, we design a novel strategy that combines a genome-wide profiling of gene expression in GRPR+ neurons with functional screening, including behavioral tests and Ca2+ imaging. Gene expression profiles between the superficial laminae with normal GRPR+ neurons and without are compared. This strategy enables us to search for the genes that are expressed in GRPR+ neurons and are functionally required for mediating nonhistaminergic itch. Three sets of experiments will be performed to test the key hypothesis that there are distinct signaling signatures for histaminergic and nonhistaminergic itch in the spinal cord. First, genome-wide profiling of gene expression in GRPR+ neurons by microarray, qRT-PCR and in situ hybridization studies will be performed. The candidate genes expressed in GRPR+ neurons will be identified. Second, the involvement of the candidate genes in acute and chronic itch will be evaluated using a siRNA knockdown approach, and the key genes for mediating nonhistaminergic itch in acute or chronic itch will be identified. Third, the relationship between the candidate genes and GRPR will be determined by examining whether the knockdown of the gene would impair intrathecal GRP-evoked scratching and would further impact scratching behavior of GRPR mutant mice. In addition, whether the candidate genes are involved in GRPR-dependent Ca2+ signaling will be examined in a heterologous system using siRNA knockdown and Ca2+ imaging. The proposed studies are highly translational because the itch mediators are identified directly from chronic itch models, and likely to be itch-specific. Our proposed studies will significantly expand the pool of the targets that may be explored for future drug discovery program.